One of this laboratory's long term goals is to contribute to a better understanding of prolactin's actions and signaling mechanisms. To support this goal we have developed a novel mouse model in which the prolactin (PRL) gene has been disrupted and propose here to study the regulation of mammary gland function using these mice. PRL actions in mammary gland growth and breast cancer remain controversial. The discovery that PRL is secreted locally in the mammary gland raises new controversies about PRL as a tumor cell growth factor. PRL knockout (PRLKO) mice provide a novel model system with which to clarify the role of PRL in breast cancer. The overall goal is to test the hypothesis that PRL is a differentiative signal that drives mammary gland maturation in balance with the proliferative effects of estradiol. This hypothesis makes specific predictions that will be challenged through experiments in four specific aims. Aim 1 will test the prediction that PRL treatment of PRLKO mice promotes differentiative phenotypic changes in the mammary gland. Differentiation-associated markers are predicted to be deficient in the mammary gland of virgin PRLKO mice. To test this measurements will be made of morphological, proliferative, apoptotic and immunocytochemical changes in the mammy gland epithelium and extracellular matrix of mice treated, or not, with PRL. The expression levels and activation of Jak2, Stat5a/b, Stat1 will be compared in PRLKO and normal mice. Aim 2 will use PRLKO mice to test the interactions between ovarian steroids and PRL by assaying changes in proliferation and differentiation markers in the mammary gland. Ovariectomized mice, treated or not with estradiol, progesterone and PRL will be used for these studies. Aim 3 will test the hypothesis that local PRL contributes to phenotypic differentiation of the mammary gland. Mammary gland tissues from normal mice will be transplanted into PRLKO mice to determine if local PRL alters epithelial function. Reciprocal transplantations will be done to determine if PRLKO mammary tissue can be stimulated to normal development through puberty, pregnancy and lactation. Aim 4 will test the effects of PRL on initiation and progression of mammary gland tumors in PRLKO mice. PRL may have different effects on tumor initiation and tumor growth because of its dual actions as a differentiation and trophic hormone. PRL will be given before and/or after carcinogen (DMBA) exposure to determine its effects on tumorigenesis. These experiments are essential for understanding PRL actions in mammary gland and breast cancer.